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Antimicrobial Agents and Chemotherapy, April 2005, p. 1381-1390, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1381-1390.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors

Victoria Chung,1* Anthony R. Carroll,1 Norman M. Gray,2 Nigel R. Parry,1 Pia A. Thommes,1 K. Claire Viner,1 and Eric A. D'Souza3

Departments of Virology,1 Computational and Structural Sciences,2 Discovery Medicine, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom3

Received 30 June 2004/ Returned for modification 10 September 2004/ Accepted 17 December 2004

A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

* Corresponding author. Mailing address: Department of Virology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom. Phone: 44(0)1438 763910. Fax: 44(0)1438 764263. E-mail: Vicky.2.chung{at}gsk.com.

Antimicrobial Agents and Chemotherapy, April 2005, p. 1381-1390, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1381-1390.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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